Evaluation of pro- and anti-inflammatory interleukins in the mammary gland of goats experimentally infected with Staphylococcus chromogenes.

The aim of this work was to evaluate the relative gene expression levels of the cytokines IL- 1B, IL-8, IL-12, IFN-γ, IL-4, IL-10 and TGF-ß in somatic milk cells of French Alpine breed, anestrous goats that were experimentally infected in the left mammary gland with Staphylococcus chromogenes during the lactation peak. Milk samples were obtained from both glands for 21 consecutive days post infection. Total RNA was extracted, and real-time PCR was conducted using primers specific to each cytokine. The relative RNA expression of the evaluated cytokines was determined by the comparative method 2-ΔΔCT, using milk from the right gland of the goats as a reference (control) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. According to the Wilcoxon test results, IL-1B and IL-12 expression levels showed significant differences compared to those in the control group (p⟨0.05) from 24 hours post infection until the end of lactation; on day three, IL1ß, IL8, IL12 and TGF-ß had a statistically significant change in expression with respect to those in the control group (p⟨0.05); closer to the end of the lactation period, there is no overexpression of the anti-inflammatory interleukins (IL-4 and TGF-ß) which may reflect the effort of the host immune system to eradicate the microorganism from the mammary gland.

Critical review of ropinirole and pramipexole - putative dopamine D(3)-receptor selective agonists - for the treatment of RLS.

Ropinirole hydrochloride (REQUIP, ADARTREL) and pramipexole dihydrochloride (MIRAPEX, SIFROL) are two putative dopamine D(3) receptor subtype-selective agonists recently approved by the FDA for the treatment of 'restless legs syndrome' (RLS). RLS is a difficult to define condition that is possibly more prevalent than previously thought. Direct-to-consumer advertising has raised public and professional awareness of RLS, but questions, even skepticism about the very existence of the condition, persist. The drugs have adverse effects that can negatively impact on quality of life and thus, as true for all drugs, require consideration of the benefit : risk ratio. We review the definition, diagnostic criteria, pathophysiology, and treatment of RLS, and assess the clinical and preclinical evidence for a pharmacologic rationale for D(3) agonism in general and of the claimed D(3) selectivity of ropinirole and pramipexole in particular.

Histological differences in the adrenal glands and cortisol levels of suckling dairy goat kids in enriched and non-enriched environments.

Intensive production systems can negatively affect the welfare of goats. Environmental enrichment techniques can be used to reduce stress. The aim of this study was analyze the effect of environmental enrichment on the histological characteristics of adrenal glands, cortisol levels and weekly weight gain of suckling Alpine French male kids under confinement. A randomised design was used to test the effect of the treatment. The animals (n=20) were randomly assigned to two treatments (enriched and non-enriched) with two replicates each. Enrichment elements included elevated sacks of henequen, trunks, tires and coconuts. The cortisol levels were measured weekly. Male kids were euthanized when their weight reached 10kg, and immediately after euthanasia, samples of the adrenal glands were collected. The adrenal glands were weighed and measured, and histological sections were taken and fixed. Four hundred cells were measured from each animal, with two blind measurements taken from each sample. There were no significant differences between experimental groups (P>0.05) in the weigh, size, the area of cells from the glomerulosa and fasciculata areas of the adrenal glands, the cortisol levels and weekly weight gain. However, there were histological differences between the glomerular and fascicular zones of the left and right adrenal glands of the different groups (P<0.05). These findings suggest that adrenal glands of animals in non-enriched environment, contained histological changes, suggestive of increased activity. We suggest testing adrenal histology as an indicator of stress and recommend the use of environmental enrichment as a means to reduce stress.

Gene localization in a family with X-linked syndromal mental retardation (Prieto syndrome).

A mapping study was performed on a 3-generation Spanish family with X-linked syndromal mental retardation. Affected males have a typical facial appearance, ear malformations, abnormal growth of teeth, clinodactyly, dimpled skin at the lower back, and patellar luxation. In pneumoencephalography a marked subcortical cerebral atrophy was evident. In the linkage studies with polymorphic DNA markers, no recombination was found between the disease locus and the loci OTC and DXS148, both assigned to Xp21.1. One or more recombinants were observed between the disease locus and loci from the distal part of Xp and the pericentromeric region. Close linkage to loci of Xq has also been excluded. The analysis of multiple informative meioses suggests that the disease locus maps between DXS255 (Xp11.22) and DXS84 (Xp21.1) on Xp.

Gene of type II autosomal dominant retinitis pigmentosa maps on the long arm of chromosome 3.

Linkage analysis has been performed on a large Australian family segregating for the autosomal dominant form of retinitis pigmentosa (ADRP). The majority of patients had no subjective symptoms of night blindness until their second decade and good visual acuity until late in life. The disease in this family has been classified as Type II ADRP according to the subdivisions provided by both Massof and Finkelstein and Fishman and colleagues. Linkage (Omax:0.08 at Zmax:4.78) is here demonstrated between the disease locus and D3S47 (a marker locus on the long arm of chromosome 3), which showed in an earlier study very close linkage without recombination to the disease locus in an Irish pedigree with a clinically more severe and early onset (Type I) ADRP.

The role of subunit interfaces for the nicotinic acetylcholine receptor's allosterism.

Cross-linking of nicotinic acetylcholine receptors, combined with binding studies and patch-clamp electrophysiology, has proven the existence of a 'pre-existing equilibrium' of functional states and the functional role of subunit interfaces, two key postulates of the allosteric model.

Prevalence of Fasciola hepatica (ELISA and fecal analysis) in ruminants from a semi-desert area in the northwest of Mexico.

The aim of the present study was to estimate the prevalence of Fasciola hepatica in cattle, goats and sheep from the municipalities of Guaymas and Cajeme, Sonora State, Mexico using the indirect ELISA and Benedeck's sedimentation tests. A total of 2,936 serum and fecal samples from 1,346 bovines, 1,199 goats and 381 sheep were analyzed. Other parameters such as breed and age were also taken into consideration. The results indicated a moderate percentage of prevalence for fasciolosis in these municipalities. In bovines, a prevalence of 11.4 +/- 0.9 was found using the sedimentation test and 24.4 +/- 1.2 for the indirect ELISA. In goats, a prevalence of 24.5 +/- 1.2 was found for the fecal analysis and 43 +/- 1.5 for the indirect ELISA. In sheep, the prevalence for both tests was 19.4 +/- 2.0 and 30.6 +/- 2.7, respectively. Results showed a greater proportion of positive animals when the ELISA test was used than when the Benedeck's sedimentation test was applied. Moderate agreement was observed between both laboratory tests for the three species of ruminants included in the study (Cohen's kappa, K; cattle K=0.527, goats K=0.541 and sheep K=0.539).

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking.

Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an "unconventional allosteric protein" which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3'-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly "freezes" the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states.

The in vitro and in vivo phosphotyrosine map of activated MuSK.

The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in neuromuscular synapse formation. Activation of MuSK is induced by agrin leading to clustering of several proteins, including acetylcholine receptors, at synaptic sites. In a first step to elucidate the signal transduction cascade following MuSK activation and leading to clustering of synaptic proteins, we sought to identify the tyrosine residues that are phosphorylated in activated MuSK. We mapped the tyrosine residues that are phosphorylated in vitro and in vivo using methods that provide high sensitivity and do not require radioactive tracers. We expressed MuSK in insect cells by using a baculovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an in vitro kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In addition, we isolated MuSK from Torpedo electric organ and used nanoelectrospray tandem mass spectrometry and parent ion scanning to identify the tyrosine residues that are phosphorylated in activated, endogenous MuSK in vivo. We found that six of the nineteen intracellular tyrosine residues in MuSK are phosphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyrosines within the activation loop (Y750, Y754, and Y755), a tyrosine near the beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C-terminal lobe of the kinase domain. Our biochemical data are consistent with results from functional experiments and establish a good correlation between tyrosine residues that are phosphorylated in activated MuSK and tyrosines that are required for MuSK signaling.

The accessible surface of the nicotinic acetylcholine receptor. Identification by chemical modification and cross-linking with 14C-dimethyl suberimidate.

To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.

No evidence of linkage between the locus for autosomal dominant retinitis pigmentosa and D3S47 (C17) in three Australian families.

A linkage analysis has been performed on three Australian families segregating for autosomal dominant retinitis pigmentosa (ADRP). No evidence of linkage has been found in any of the pedigrees studied between the locus D3S47 and the gene for ADRP. The D3S47 locus was found to show very close linkage with the ADRP gene in a large Irish pedigree. Our study together with a similar report on a British family indicates that there is genetic heterogeneity in this disease.